Neural stem cells

ABSTRACT

A homogenous, symmetrically dividing population of adherent neural stem cells is obtained from ES cells or foetal or adult brain isolates, using an activator of a signalling pathway downstream of a receptor of the EGF receptor family, optionally in combination with an activator of a signalling pathway downstream of an FGF receptor. The neural stem cell population is highly pure and retains the ability to differentiate into neurons after in excess of 100 passages.

The present invention relates to neural stem cells and to cultureconditions and methods of culturing neural stem cells (NS cells or NSCs)in order to promote symmetrical division and self renewal of the stemcells. Compositions, cell populations, cell lines and single neural stemcells are also provided.

While neural stem cells have been purported to be isolated in vitro froma variety of sources it has not, to date, been possible to expand thecells in large-scale culture in a symmetrically-dividing,undifferentiated state. The long-term expansion of the cells in thisstate would be highly desirable, both from an experimental andtherapeutic point of view. Having pure neural stem cell populationswould enable directed differentiation into the three cell types ofneurons, astrocytes and oligodendrocytes.

One known method of culturing neural stem cells, albeit as a minorcomponent in a heterogeneous population of cells, is the neurospheresystem. This system involves the serial passaging of aggregates ofheterogeneous cells, of which only a tiny fraction are true neural stemcells. The majority of cells in neurospheres are committed progenitors.

There are many reports of the heterogeneity and instability ofneurospheres, and their limited capacity to generate neurons (e.g.Morshead et al., 2002). Rappa et al. Neuroscience 2003 report a methodfor transfecting neurosphere cells by plating them on fibronectin for1-7 days then reforming neurospheres but provides no significantcharacterization of the stem cells, if any, within neurospheres.

Suslov et al 2002. provide clear data that the neurosphere system is tooheterogeneous to enable meaningful characterization of any stem cellswithin. The major conclusions are that not only is there heterogeneitywithin neurospheres—“that exhibit intra-clonal neural cell-lineagediversity i.e. they contain, in addition to NSCs, neuronal and glialprogenitors in different states of differentiation”—but also that largedifferences exist in gene expression profiles between different clonalneurosphere lines. Suslov's gene expression profiling does notconstitute a definition of stem cells within neurospheres as explicitlyconfirmed in the paper:—“the molecular phenotypes that were obtainedindicate that clonogenic NSCs in our system are heterogeneous, withsubsets reflecting distinct neural developmental commitments”.

Much literature in this field describes the properties of glial cells.Skogh et al. MCN 2001 describe cultures of glial cells that express GFAPand stain variably for RC2 and nestin—note that mouse GFAP is notexpressed in radial glia in vivo (Rakic, 2003). They report that theircells do not express Pax6, the hallmark of neurogenic radial glia(Malatesta et al., 2001, 2003), and they examine no other radial gliamarkers. They had heterogeneous cultures and carried out no clonalanalyses.

Other accounts of radial glia in vitro consider them as differentiationintermediates or terminal products. Thus Bibel et al (Nat Neurosci,2004) report the generation of neurons from ES cells via a transientradial glia-like cell, and Gregg and Weiss (J Neurosci, 23: 11587-601,2003; U.S. patent publication 2003/0032181) describe “differentiation”of neurosphere cells into radial glia. Hartfuss et al (Dev Biol, 2001)show that radial glia are present in neurospheres.

Gregg and Weiss found that radial glia are differentiation products ofneurospheres, concluding that “These results suggest that neural stemcells can give rise to RGCs [radial glia cells] and that RGC-guidedmigration can be recapitulated in the adult CNS”. The prevailing view isthat radial glia are one of the differentiated cell types arising fromneurospheres.

Liour and Yu (Glia, 2003) show that radial glia cell differentiation canbe obtained from ES cells, but as with other reports in this field donot report expansion of neurogenic radial glia.

Gobbel et al. Brain Res 2003 report propagation of multipotent rat cells“as spherical clusters of cells that grew loosely attached to thesubstrate”, i.e. not as uniform monolayers. Further, the abstract ofthis paper concludes “these cells produce post-mitotic cells inapproximately two of three divisions, thus making expansion difficult”.There is extensive gliogenic differentiation in their cultures, but theauthors find “relatively few” (<3%) neurons. They provide no molecularphenotype of their cells.

WO 01/30981 describes cultures of cells that can differentiate intoneurons. But these cells are positive for GFAP and nestin, are describedas astroglial cells and are a relatively impure, mixed population.

The concept that stem cells require specific cellular microenvironments,or niches, is an orthodoxy in stem cell biology. It is known, though notalways acknowledged, that in neurospheres stem cells constitute only asmall fraction of the total cells amongst progenitors, blast cells andimmature differentiated progeny. This aggregation of mixed cell typesmay constitute a niche for the small proportion of cells within that maybe stem cells.

There are no reports in the literature of deriving homogenouspopulations of neural stem cells from ES cells, only of derivingneuroepithelial precursor cells that may be expanded transiently beforebecoming glial-restricted (e.g. Brustle et al., Science 1999).

Hence a number of problems exist in the art.

All reports acknowledge that their cultures are heterogeneous. It hasnot been possible to use the neurosphere system to maintain large scalecultures of neural stem cells in a symmetrically-dividing andundifferentiated state. Other attempts to culture large numbers ofneural stem cells have not succeeded beyond 5-20 passages, and have alsobeen hindered by a high tendency of the cells to differentiate. Anexception is work on adult rat hippocampal stem cells, but these arekaryotypically abnormal and form neurons at low efficiency.

Whilst transient neural developmental precursors are known, permanent ornear-permanent self-renewing stem cells have not been isolated andpurified.

It is desired to derive neural stem cells from ES cells, and thenmaintain these neural stem cells in pure cultures, but this is hithertonot possible. It is also desired to derive neural stem cells from EScells for transplantation but the persistence of ES and other non-neuralcells in known ES-derived cell populations gives rise to tumours inrecipient animals. It is further desired to obtain pure neural stempopulations from foetal and post-natal CNS.

A complete understanding of the molecular and cellular eventscontrolling the behaviour of neural stem cells is essential, not only asa route for understanding embryogenesis, but also as a framework uponwhich neural stem cells can be isolated, expanded and controlled forfuture therapeutic applications. The culturing methods for neural stemcells known in the art are, for reasons described above, unsuitable foruse in such investigations and therapeutic applications. It is thereforedesirable to develop methods and conditions for culturing largequantities of neural stem cells that allow the cells to be maintained ina symmetrically-dividing, self-renewing state. It is particularlydesirable to have defined culture media, which meet the aboverequirements as the use of defined media is highly desirable in aclinical setting.

The present invention solves one or more of the above-mentionedproblems.

In more detail, the present invention provides a method of promoting thesymmetrical division of neural stem (NS) cells, comprising culturingsaid cells in a medium containing:

-   -   (a) an activator of a signalling pathway downstream from a        receptor of the EGF family; and    -   (b) an activator of a signalling pathway downstream from an FGF        receptor.

In preferred embodiments, cells are cultured in the absence of serum,e.g. in medium that is free of serum and free of serum extract. It isfurther preferred that cells are cultured attached to a substrate,otherwise referred to as in an adherent culture. It is also preferredthat culture medium contains insulin or another agonist of an insulinreceptor on the cells.

In the context of the invention, it will be appreciated that the term“promoting” includes the maintenance of the neural stem cells in asymmetrically-dividing state.

According to another aspect of the present invention there is providedculture media that supports and preferably promotes the self-renewal andsymmetrical division of neural stem cells in an undifferentiated statefor many passages. The media substantially prevents the asymmetricaldivision and differentiation of neural stem cells.

The invention further provides methods of obtaining neural stem cells,and specifically as set out in the protocols herein, and additionallyprovides cells obtained by such methods.

The invention also provides neural cell populations, compositions, celllines, clonogenic cell lines, and single neural stem cells, whichcomprise self-renewing, symmetrically-dividing neural stem cells.

Further aspects of the invention provide methods for promoting thedifferentiation of ES cells to neural stem cells, and methods formaintaining the neural stem cells obtained in a self-renewing,symmetrically-dividing, substantially undifferentiated state.

DEFINITION SECTION

“Neural Stem Cells”

The term “neural stem cell”, as used in the present specification,describes a cell that is capable of undergoing greater than 20-30 celldivisions whilst maintaining the potency to generate both neurons andglia. Preferably, said cells are capable of undergoing greater than 40,more preferably greater than 50, most preferably unlimited such celldivisions.

Neural stem cells are capable of dividing either symmetrically, orasymmetrically. When dividing symmetrically, the neural stem celldivides to form two daughter neural stem cells or two committedprogenitors, though unless otherwise specified symmetrical divisionrefers herein to symmetrical self renewal; when dividing asymmetrically,the neural stem cell divides to form one daughter neural stem cell, andone committed progenitor (e.g. either a neuron or a glial progenitor).

Neural stem cells of the present invention can be described as radialglia, and have been shown to express at least one (and preferably all)of the radial glia markers RC2, 3CB2, GLAST, BLBP and Pax6. Preferably,the neural stem cells of the invention express RC2, 3CB2 and GLAST. Morepreferably, the cells express RC2, 3CB2, GLAST, and at least one of BLBPor Pax-6. Neural stem cells of the invention can also be characterizedin that they are positive for the expression of at least one (preferablyall) of the neural precursor markers nestin or vimentin, the LewisXantigen, Musashi-1 or prominin, and negative for the expression of atleast one (preferably both) of Oct-4 or Nanog (see also Example 1-3).

The neural stem cells of the invention are by definition multipotent,i.e. they are capable of differentiating into a number of neural celltypes (e.g. neurons/glia). Examples below confirm that neural stem cellscultured according to the methods of the invention retain their potencyand are able to differentiate into all expected cell types.

“Sources of Neural Stem Cells”

It is possible to derive neural stem cells of the invention from a widevariety of sources. For example, neural stem cells can be deriveddirectly from embryos, from adult tissue, from foetal tissue, or fromembryonic stem (ES) cells (either wild-type or genetically modified EScells). Preferably, the neural stem cells of the invention are derivedfrom mouse or human ES cells, or are derived from mouse or human foetalcells.

Neural stem cells of the invention can be derived from, interalia,humans, primates, rodents, and birds. Preferably, the neural stem cellsare derived from mammals, especially mice, rats and humans.

“EGF Receptor Family”

The term “EGF receptor family”, as used in the present specification,describes a family of receptors (usually homodimeric or heterodimericreceptors) which can be activated by a family of EGF signalling factors.The receptors are made up of a family of four highly homologoustransmembrane glycoproteins: ErbB-1 (also known as EGF-R), ErbB-2,ErbB-3 and ErbB4.

Reference to an “EGF receptor” includes any monomeric or dimericreceptor complex of the EGF receptor family.

Each receptor has an extracellular ligand binding domain, a singlehydrophobic membrane-spanning domain and a cytoplasmic tyrosine kinasedomain that is responsible for signal transduction through Type-1receptor tyrosine kinase activity. Ligand binding to any of thereceptors results in receptor dimerisation and autophosphorylation,followed by phosphorylation of a number of cellular substrates leadingto an array of biological effects. The receptor dimerisation may betriggered by various stimuli, including receptor ligands and toxicenvironmental stimuli such as UV radiation. Each dimeric receptorinitiates a distinct signalling pathway by recruiting differentSH2-containing effector proteins. For example, the EGF-R dimer cancomplex with the adaptor protein Grb, coupled to a guanine nucleotidereleasing factor, SOS. The Grb-SOS complex can either bind directly tophosphotyrosine sites in the receptor, or indirectly through Shc. Theseprotein interactions bring SOS in close proximity to Ras, allowing forRas activation. This subsequently activates the ERK and JNK signallingpathway that in turn activate transcription factors, such as c-fos, AP-1and Elk-1, that regulate gene expression. The EGF receptors can alsoactivate the PLCγ signalling pathway.

“FGF Receptor”

The term “FGF receptor”, as used in the present specification, describesany member of the family of transmembrane FGF receptor tyrosine kinases.There are four main isotypes of these receptors, FGFR1, 2, 3 and 4, andthey are known to act in close association with the heparin and heparansulphate (HS) systems. Reference to an FGF receptor includes anymonomeric or dimeric (homo or heterodimer) complex of the FGF receptorfamily.

Cellular signalling pathways associated with FGF receptors include theMAP kinase pathway, and the PLCγ pathway.

” Culture Media”

Culture media used in the present invention preferably comprise a basalmedium, optionally supplemented with additional components.

Basal medium is a medium that supplies essential sources of carbonand/or vitamins and/or minerals for the neural stem cells. The basalmedium is generally free of protein and incapable on its own ofsupporting self-renewal/symmetrical division of neural stem cells.

Preferably, culture media used in the invention do not contain anycomponents which are undefined (e.g. serum and/or feeder cells), that isto say components whose content is unknown or which may containundefined or varying factors that are unspecified. An advantage of usingfully defined media, free of serum and free of serum extracts, is thatefficient and consistent protocols for culture and subsequentmanipulation of neural stem cells can be derived.

“Culture Surfaces”

Typical surfaces for culture of the neural stem cells in all aspects ofthe invention are culture surfaces recognized in this field as usefulfor cell culture, and these include surfaces of plastics, metal,composites, though commonly a surface such as a plastic tissue cultureplate, widely commercially available, is used. Such plates are often afew centimetres in diameter. For scale up, this type of plate can beused at much larger diameters and many repeat plate units used.

The culture surface may further comprise a cell adhesion protein,usually coated onto the surface. Receptors or other molecules present onthe stem cells bind to the protein or other cell culture substrate andthis promotes adhesion to the surface and promotes growth. Gelatincoated plates are particularly preferred.

The present invention is based on the observation that culturing neuralstem cells attached to substrates in media comprising agonists of theEGF receptor, or agonists of both the EGF and FGF-2 receptors, promotesthe unlimited, symmetrical division of the stem cells, and substantiallyprevents their differentiation into neurons/glia.

The various aspects of the invention are now discussed in detail.

A first aspect of the invention provides a method of promoting thesymmetrical division of neural stem (NS) cells, comprising culturingsaid cells in a medium containing:

-   -   (a) an activator of a signalling pathway downstream from a        receptor of the EGF family; and    -   (b) an activator of a signalling pathway downstream from an FGF        receptor.

Another method of the invention, for obtaining neural stem cellscomprises:

(1) obtaining a mixed population of cells containing a neural stem cell;

(2) replating the cells in medium comprising (a) an activator of asignalling pathway downstream from a receptor of the EGF family; and (b)an activator of a signalling pathway downstream from an FGF receptor;

(3) culturing the cells;

(4) harvesting aggregates of cells;

(5) replating the cells in medium comprising (a) an activator of asignalling pathway downstream from a receptor of the EGF family; and (b)an activator of a signalling pathway downstream from an FGF receptor;

(6) culturing the cells;

(7) replating cells as single cells in medium containing (a) anactivator of a signalling pathway downstream from a receptor of the EGFfamily; and (b) an activator of a signalling pathway downstream from anFGF receptor.

The method can be supplemented by selecting for cells that express aneural stem cell specific marker, for example by linking expression ofsuch a marker to a promoter preferentially active in neural stem cellscompared to its expression in differentiated progeny thereof. Preferablythe method comprises passaging the cells at or below 65% confluence,more preferably at or below 55% confluence, especially below 50%confluence.

Other preferred features of methods of the invention are as set out inthe protocols of the invention in Example 9.

The cultures of the invention are preferably adherent cultures, i.e. thecells are attached to a substrate.

The substrate is typically a surface in a culture vessel or anotherphysical support, e.g. a culture dish, a flask, a bead or other carrier.Preferably, the substrate is coated to improve adhesion of the cells andsuitable coatings include laminin, poly-lysine, poly-ornithine andgelatin. It is also preferred that the cells are grown in a monolayerculture and not in suspension and not as balls or clusters of cells. Athigher densities, cells may begin to pile up on each other, but thecultures are essentially monolayers or begin as monolayers, attached tothe substrate.

One or more signaling pathways downstream from a receptor of the EGFfamily can preferably be activated using an agonist of a receptor of theEGF family. An agonist of a receptor of the EGF family is suitably amember of the EGF family of signaling factors, and preferably binds tothe extra cellular domain of the EGF receptor. The term “agonist” alsoembraces mimetics, fusion proteins, antibodies to or chimeras of the EGFfamily of signalling factors, and fragments, variants and derivativesthereof, capable of activating receptors of the EGF family.

The molecules that make up the EGF family of signalling factors can becharacterized in that they contain at least one EGF-like domain. Thisdomain can be defined by 6 cysteine residues that generate three peptideloops through the formation of disulphide bonds.

Specific agonists capable of acting through receptors of the EGFreceptor family, and thus of activating pathways downstream of thesereceptors, include EGF, TGF-α, amphiregulin, heparin binding-EGF,epiregulin, betacellulin, neuregulins 1-4, and Cripto-1. Preferably, theagonist is EGF itself.

One or more signalling pathways downstream from a receptor of FGF canpreferably be activated using an agonist of a receptor of FGF. Anagonist of a receptor of FGF is suitably a member of the FGF family ofsignalling factors. The term “agonist” is also intended to embracemimetics, fusion proteins, antibodies to or chimeras of the FGF familyof signalling factors, and fragments, variants and derivatives thereof,capable of activating receptors of FGF.

Preferably, the agonist of the FGF receptor is FGF-2, or laminin-FGF.

It will be appreciated that activation of a signalling pathwaydownstream of a receptor of either the EGF receptor family or of an FGFreceptor can also be effected by constitutively active receptors, or bydownstream effectors (e.g. MEK or Bcl2) of the respective signaltransduction pathways. In a particularly preferred embodiment of theinvention, the signalling pathways are activated by cell-permeable smallmolecules, which can bypass the respective receptors and activate thesignalling pathways directly. Thus, in the present invention, the term“activator” embraces all molecules capable of activating a signallingpathway downstream of receptors of the EGF family of receptors, or of anFGF receptor.

The effectiveness of the activators in the maintenance of neural stemcell cultures of the present invention is demonstrated in Examples 1-1and 1-2 below. Here, bulk and clonal populations of neural stem cellsare maintained in media comprising EGF and FGF-2 and may be passagedmany times. Moreover, there is negligible/no differentiation of theneural stem cells, as is shown by the ubiquitous presence of the neuralstem cell markers in the cell populations tested (see Example 1-3), andthey retain their potency and are able to differentiate into allexpected cell types (see Example 1-4).

Thus, the invention provides an efficient method of maintaining largepopulations of neural stem cells, in a self-renewing,symmetrically-dividing, undifferentiated state. Compositions of theinvention include compositions comprising neural stem cells, wherein theneural stem cells are in an adherent culture and at least 50%, 70% or80% of the cells in the composition are neural stem cells. Theproportion of neural stem cells is further preferably at least 90%, morepreferably at least 95%, very preferably at least 97%. Further, neuralstem cells of the invention can be passaged extensively. It is preferredthat the neural stem cells have been passaged at least 30 times, morepreferably at least 60 times, very preferably at least 90 times. Stillfurther, in a population of neural cells of the invention at least 80%,preferably at least 90%, more preferably at least 95%, of said cells aresymmetrically-dividing neural stem cells. The cells in this compostioncan be further characterized by any and all features of the invention,alone or in combination

Also provided are compositions, which comprise:

-   -   neural stem cells;    -   an activator of a signalling pathway downstream from a receptor        of the EGF family; and    -   an activator of a signalling pathway downstream from an FGF        receptor.

The compositions preferably comprise a basal medium. It is alsopreferable that at least 80%, preferably 90%, more preferably 95% of theneural stem cells in the composition are symmetrically-dividing neuralstem cells.

In both the neural cell populations and compositions described above,the neural stem cells are preferably free from exogenous geneticmaterial encoding an oncogene, i.e. that they have not been subjected toimmortalisation strategies. In specific embodiments, the neural stemcells of the populations/compositions are characterized in that they arepositive for the expression of at least one of:

-   -   the neural precursor markers nestin or vimentin;    -   Sox-2;    -   the radial glia cell specific markers RC2, 3CB2, GLAST, BLBP or        Pax-6;    -   the LewisX antigen;    -   Musashi-1; and    -   Prominin;        and are negative for the expression of at least one of:    -   Oct4, and    -   Nanog.

Preferably, the cells are positive for the expression of RC2, 3CB2 andGLAST. In other embodiments, (optionally in addition to the precedingmarker profile) the cells are positive for the expression of at leastone of BLBP, Pax-6, the neural precursor markers nestin or vimentin, theLewisX antigen, Musashi-1 or prominin. In particularly preferredembodiments (and optionally in addition to the preceding markerprofiles) the cells are negative for the expression of at least one ofOct4 or Nanog.

In further embodiments (optionally in addition to the above markerprofiles), the cells are positive for the expression of Sox-2, andnegative for the expression of Sox-1. In certaincompositions/populations comprising mouse-derived NS cells, no more than1% of the cells are positive for the expression of GFAP or βIII tubulin,thereby confirming that there is negligible differentiation of the cellsto astrocytes or neurons. In other compositions/populations, no morethan 1% of the cells are positive for the expression of markers formature astrocytes, neurons or oligodendrocytes.

The invention has also been carried out with rat cells. Thus, we havetaken cells from the rat CNS and using the methods of the inventionobtained a culture of rat neural stem cells having the properties of (i)high purity, typically in excess of 80% or 90% rat neural stem cells,and (ii) being cells a high proportion (a minimum of 50%) of which aftermany doublings, in excess of 50, in excess of 100, even in excess of 200doublings, retained the ability to form neurons and glia. In the artcells from the rat were clearly highly heterogenous and only from theadult hippocampus. We have not obtained cells from this source, butinstead from rat CNS. It is notable in this respect that the method ofthe invention has worked across all three species—rat, mouse, human. Itis generally seen that if a single cell will form a neural stem cellcolony following the methods of the invention then neurons can beobtained from cells in that colony. This is measured for example bystaining separately for (i) nuclei (i.e. staining all cells) and (ii)neurons (i.e. only neurons). By comparing the relative numbers ofstained cells the proportion of cells that form neurons can becalculated,

A second aspect of the invention provides a method for the preparationof a neural stem cell, comprising (i) culturing neural stem cellsaccording to the methods described above, thereby obtaining a culture ofneural stem cells, and (ii) isolating a neural stem cell from saidculture. Preferably, the isolated neural stem cell is one that has beenconditioned to divide in a symmetrical manner.

A number of sources of cells can be used from which to derive neuralstem cells. In one method, cells are obtained from nervous tissue of ananimal and cultured according to the invention, to obtain a culture ofsymmetrically dividing neural stem cells. The nervous tissue can be fromadult or foetal tissue. The nervous tissue can be from CNS, andsymmetrically dividing populations of neural stem cells can be obtainedfrom extracts taken from both adult and foetal CNS, from both human andmouse. The nervous tissue preferably comprises cells identified ashaving a radial glial phenotype, for example cells having this phenotypehave been identified in extracts from the cerebellum and retina, both ofwhich represent suitable further sources of cells. Neural stem cells canbe obtained from diseased nervous tissue, useful for modeling disease,e.g. cells can be obtained from brain tumours for this purpose.

An option is to use the invention to derive neural stem cells from adiseased individual then (i) use those cells for assays, or (ii) carryout genetic modification prior to transplantation of cells back intothat individual. Cells may thus be obtained from an individual with aneurodegenerative disorder, examples including Alzheimer's andParkinson's diseases; or with a brain tumour.

Another method of obtaining a neural stem cell comprises:

(i) obtaining a multipotent or pluripotent stem cell capable ofdifferentiating into a neural stem cell,

(ii) culturing the cell of (i) in medium which is non-permissive forpluripotent cells and in the presence of (a) an agonist of a signallingpathway downstream of a receptor of the EGF family and (b) an agonist ofa signalling pathway downstream of a receptor of the FGF family.

In use, potentially contaminating ES cells are removed by this method,or at least substantially reduced in number, giving a purer culture andone less likely to give rise on transplantation to teratomas.

The stem cell is suitably a pluripotent stem cell, especially an ES orEG cell. The method preferably comprises culturing the cell in thepresence of an agonist of the EGF receptor and an agonist of the FGFreceptor.

Using NS cells of the invention, we have obtained astrocytes by removingEGF and FGF and adding serum or BMP4, we have obtained neurons by takingaway EGF, then taking away FGF after an interval of about a week andplating the cells on laminin and we have obtained oligodendrocytes bytaking away both EGF and FGF. Generally, all methods of the inventionused to generate a neural stem cell optionally comprise a further stepof differentiating the neural stem cell into a neuron, an astrocyte oran oligodendrocyte, and then further optionally using the neuron,astrocyte or oligodendrocyte e.g. in an assay, for transplantation orotherwise.

Once a neural stem cell culture has been isolated, it can then be usedto establish a neural stem cell line. The establishment of such a cellline preferably includes the steps of:

-   -   (a) obtaining a single neural stem cell,    -   (b) culturing the neural stem cell according to any of the        methods described previously,        and thereby obtaining a clonal population of neural stem cells.

In specific embodiments, the single neural stem cell used to establishthe cell line is a symmetrically-dividing neural stem cell, or a neuralstem cell that has been conditioned to divide in a symmetrical manner.Preferably, the single neural stem cell is obtained according to themethod described above for preparing a neural stem cell.

In preferred embodiments, the neural stem cell line obtained by theabove method is a clonal population of neural stem cells, i.e. whereinall the cells are progeny of a single neural stem cell.

Single neural stem cells and cell lines (optionally obtainable by themethods described above) also form part of the invention. In a firstembodiment, cell lines are provided wherein the cells are maintained inthe presence of an activator of a signalling pathway downstream of areceptor of the EGF receptor family, and an activator of a signallingpathway downstream of an FGF receptor. In other embodiments, the singleneural stem cells and the cells of the cell lines are characterized inthat they are positive for the expression of at least one of:

-   -   the neural precursor markers nestin or vimentin;

Sox-2;

the radial cell specific markers RC2, 3CB2, GLAST, BLBP or Pax-6;

-   -   the LewisX antigen;    -   Musashi-1; and    -   Prominin;        and they are negative for the expression of at least one of:    -   Oct4, and    -   Nanog.

Preferably, the cells are positive for the expression of RC2, 3CB2 andGLAST. In other embodiments, (optionally in addition to the precedingmarker profile) the cells are positive for the expression of at leastone of BLBP, Pax-6, the neural precursor markers nestin or vimentin, theLewisX antigen, Musashi-1 or prominin. In particularly preferredembodiments, (and optionally in addition to the preceding markerprofiles) the cells are negative for the expression of at least one ofOct4 or Nanog.

In other specific embodiments (optionally in addition to the abovemarker profiles), the cells are positive for the expression of Sox-2,and negative for the expression of Sox-1. In certain mouse-derived celllines, no more than 1% of the cells are positive for the expression ofGFAP or β III tubulin. In other (e.g. human) cell lines, no more than 1%of the cells are positive for the expression of markers for matureastrocytes, neurons or oligodendrocytes.

It is preferable that both the single neural stem cells, and the cellsof the cell lines, are free from exogenous genetic material encoding anoncogene, i.e. that they have not been subjected to immortalisationstrategies. It is also preferable that the cell lines described areneural stem cell lines.

In a third aspect, the invention provides a method of obtaining andmaintaining a transfected population of symmetrically-dividing neuralstem cells, comprising:

-   -   (a) transfecting ES cells with a construct encoding a selectable        marker A, wherein in use said selectable marker is expressed        under the control of a    -   neural precursor cell-specific promoter;    -   (b) promoting the differentiation of the ES cells into neural        precursor cells;    -   (c) selecting for neural precursor cells that express the        selectable marker A; and    -   (d) culturing the selected cells according to any of the methods        previously described.

The selectable marker A may encode antibiotic resistance, a cell surfacemarker or another selectable marker as described e.g. in EP-A-0695351.The neural precursor cell-specific promoter may be selected from thegroup consisting of Sox-1, Sox-2, Sox-3, BLBP and nestin neuralenhancer. Further details of this selection strategy are provided inExample 1-1.

The neural stem cells of the invention (i.e. single neural stem cells,and neural stem cells present in the compositions, cell lines andpopulations of the invention) can be genetically modified. There istherefore provided a method of transfecting the neural stem cells of theinvention comprising:

-   -   (a) transfecting the neural stem cells with a construct encoding        a selectable marker B and a polypeptide; and    -   (b) selecting for neural stem cells that express the selectable        marker B.

The selectable marker B may encode antibiotic resistance or a cellsurface marker, and may be the same as or different from selectablemarker A. Suitable transfection methods are known ones, includingelectroporation, lipofection, necleofection and retro-and lenti-viraltransfection.

The genetically modified neural stem cells also form part of theinvention, and the invention therefore provides a single neural stemcell, or neural stem cells as present in the compositions, populationsor cell lines of the invention, further comprising the construct. Itwill be appreciated that such neural stem cells may already compriseselectable marker A, in addition to selectable marker B.

As mentioned previously, the methods of the invention are suitable foruse with neural stem cells derived from any source. In one particularembodiment, the invention provides a method for obtaining neural stemcells from a source of ES cells. According to the method,differentiation of ES cells into neural stem cells is promoted byconverting ES cells into neural progenitors, e.g. by culture in amonolayer or embryoid body differentiation, and then culture of neuralprogenitors in NSA medium. In addition, it is contemplated that the NSAmedium may comprise supplemental components, such as supplementalglucose and HEPES. As an alternative, media (preferably basal media)supplemented with glucose and HEPES can also be used to promote thisdifferentiation.

In the presence of NSA and/or glucose and HEPES, the conditions are suchthat the propagation of neural stem cells is favoured, with the addedadvantage that any non-neural cells present in the culturepreferentially die. This results in a substantially pure culture ofneural stem cells (e.g. at least 80%, preferably 90%, more preferably95% of all cells present). Example 1-5 provides further details of thismethod.

In a preferred embodiment, the method of Example 1-5 may be used toprepare a population of symmetrically-dividing neural stem cells, whichare subsequently maintained using any of the culturing methods of theinvention described above.

The method of Example 1-5 can also be used to assay for the effect offactors on the differentiation of ES cells into neural stem cells. In apreferred assay embodiment, ES cells are cultured using the methoddescribed in Example 1-5, in the presence of the factor to be tested.The effect of the factor can be assessed by, for example, determiningthe marker profile of the resultant cells, i.e. to show whether thecells have a similar marker profile to the cells of the invention, orwhether the ES marker profile is maintained. The factors tested may beeither inductive or blocking factors.

There is much interest in the use of neural stem cells in the treatmentof neurological and neurodegenerative diseases, and brain injuries; inparticular in the treatment of diseases such as Parkinson's andAlzheimer's disease, multiple sclerosis and amyotrophic lateralsclerosis. The methods, compositions, cell populations, cell lines andsingle cells of the present invention are all capable of being used insuch treatment, as well as in the manufacture of preparations for suchtreatment. Particular neurological/neurodegenerative diseases that canbe treated using the invention include:—Parkinson's disease, motorneuron disease, stroke, multiple sclerosis, and Huntington's disease.

In a fifth aspect, therefore, the invention provides for the use of thecell lines, neural cell populations, single neural cells andcompositions described above for cell therapy and for the manufacture ofa preparation for the treatment of neurodegenerative diseases and braininjuries. Such preparations may be formulated in phosphate bufferedsaline (PBS).

Methods of treatment of the diseases listed above can comprise thetransplantation of single cells, cell lines, compositions, or cellpopulations of the invention into a patient. Preferably, the patient ismammalian, more preferably the patient is human. Such transplantationhas been shown to be successful in both embryonic and adult CNS and isdescribed in more detail in Example 1-6.

The cells of the invention, and in particular the cell lines, can beused to assay the effect of inductive or blocking factors on thedifferentiation of neural stem cells. Such an assay may comprisecontacting a neural stem cell of the invention (i.e. as present in thecompositions, cell lines, and populations, or a single neural stem cell)with the factor to be tested. The effect of the factor on thedifferentiation of the neural stem cell can be suitably assessed bydetermining the marker profile of the resultant cells, i.e. to showwhether the cells have a similar marker profile to the cells of theinvention, or whether these markers have been lost. The cells of theinvention are also suitable for assaying pharmaceuticals.

To assess the proportion of cells that will form neurons or glia, cellsare propagated clonally; cells are plated individually and not all cellsform clonal colonies but of those which do generally in excess of 50%will make neurons (under the appropriate protocol) or glia (again, underthe appropriate protocol). The cells are distinguished from the art aswhile the art alleges identification of and even propagation of cellswhich have neural stem cell properties the populations were highlyheterogenous. This is an important distinction as impure populationsinclude cells which provide signalling to remaining neural stem cellswhich stimulates differentiation and further reduction of the proportionof neural stem cells. While the prior art methods can generateneurospheres from individual cells the invention enables generation ofsubstantially pure populations from individual cells.

In particular embodiments of the invention, described in more detail inan example below, all NS cell colonies produce 15% or greater,preferably at least 20%, more preferably about 20 to 30% TuJ positiveneurons. In higher density cultures we have counted TuJ positive cells.35% of LC1 NS cells acquired neuronal morphology and expressed betaIII-tubulin after 115 passages, longer than one year of continuousculture. These cells retained a diploid karyotype at this stage. LC1 isan uncloned population. Therefore the data indicate that there wasminimal selective pressure for glial-restricted precursors or forgenetic transformation.

Cells of the invention can be grown without co-culture with heterologouscells and without undefined conditioned media, extracellular matrixfractions or serum, a feature not previously shown for any stem celltype other than ES cells. Hence following the invention, neural stemcells are preferably grown in homogenous culture in defined conditions.Even a single NSC isolated in a microwell can expand, indicating minimaldependence on extracellular signals other than EGF and FGF and thatessential extrinsic self-renewal signals for the NSCs can be reduced toEGF and FGF in vitro.

In further embodiments illustrated in an example in more detail,substantially every colony, that is at least 90%, preferably at least95%, more preferably at least 97% of colonies that develop from platingsingle cells (i) show identical homogenous expression of neuralprecursor markers and absence of differentiation markers in FGF plusEGF, and (ii) generate neurons on growth factor withdrawal. In aspecific example, every colony had these properties. This is evidencefor symmetrical self-renewal. Furthermore, data from colony assays onnon-cloned NS cell cultures were comparable with data from the clonalNS5 line. Serial formation of expandable undifferentiated colonies fromNS5 shows that the clonogenic cells are stem cells and that theirnumbers expand in proportion with the NS population. Formally anincrease in stem cell numbers can occur via two mechanisms: de novogeneration or symmetrical self-renewal divisions. The former requires apre-stem cell source i.e. a pluripotent cell or a foetal anlage, neitherof which are present in the NS cell cultures herein, so symmetricalself-renewal must be ongoing in the NS cell cultures.

Specific NS cells, obtained in an example below, express Sox2, Pax6,Emx2, Olig1, Olig2, Nestin, BLBP, GLAST, Vimentin, and areimmunoreactive for RC2, 3CB2, SSEA-1, and Prominin. Preferably they donot express Sox1 and are negative for GFAP and neuronal antigens. TheseNS cells lack pluripotency markers and markers of other germ layers. Wecontend that neural stem cell character is masked during CNS developmentand only truly demonstrable ex vivo, as is the case with ES cells. Thefinding that Sox1 expression was not maintained in the NS cells is new,unpredicted, and indicates that continuous lineage selection using Sox1would not be productive.

In further embodiments of the invention NS cells, exemplified by EScell-derived uncloned (CGR8-NS) and cloned (NS5) NS lines and foetalcortex-derived uncloned (Cor1) and cloned (Cor1-3) NS lines, showexpression of radial glia markers 3CB2, BLBP, GLAST, Nestin, RC2 andVimentin in culture. Expression of GFAP is preferably in fewer than 10%,more preferably fewer than 5% especially in fewer than 2% of cells inany of these cultures. In a specific example GFAP expression was seen infewer than 1% of cells. We have also shown uniform expression of nestinand RC2 with absence of GFAP and βIII-tubulin in ES cell derived NScells after 115 passages indicating that the NS cell phenotype isstable. Preferred cells of the invention are differentiated from EScells and (1) continue to express nestin and RC2 and (2) continue not toexpress GFAP and βIII-tubulin after 30 passages, preferably after 60passages, more preferably after 100 passages. Non-expression of GFAP andbeta III-tubulin means expression is seen in fewer than 5% of cells,preferably fewer than 2% of cells.

We have carried out analysis via RT-PCR which confirmed absence ofpluripotent, mesodermal, or endodermal specific transcription factors inspecific NS cells. We have also carried out Affymetrix (RTM) expressionprofiling that confirms the absence of lineage inappropriate transcriptsand demonstrates consistent expression of neural and radial glia markersin three different NS cell cultures (ES derived, foetal cortex-derived,and clonal foetal cortex-derived).

Obtaining NS cells by differentiation from ES cells preferably comprisesmaintaining the cells in continuous adherent culture, and morepreferably omits a step of forming a neurosphere. However, obtaining NScells by differentiation of cells from primary cell isolates from foetalor adult brain optionally comprises (i) first forming a suspensionaggregate or a neurosphere, and (ii) subsequently maintaining the cellsin adherent culture. We have found in examples that after a few days theaggregates can be attached to gelatin-coated plastic and NS cells willthen grow out.

We determined the proportion of neurons and astrocytes generated atearly and late passages of cells of the invention, specifically for theLC1 NS cells at passages 8 and 115. The latter culture represents 12months of expansion with a doubling period of 24 hours. There is amodest decline in the number of neurons obtained at late passage, butthis still totals>35%. The NS5 clonal line, independently derived from46C ES cells, shows a similar level of neuronal differentiationefficiency at passage 30. Generation of GFAP immunoreactive astrocytesapproached 100% at both time points for the LC1 NS cells and also forNS5 cells. Furthermore, the in vivo data, which show extensivedifferentiation of both neurons and astrocytes, were obtained using LC1NS cells grown for multiple passages, then transduced with GFPlentivirus and expanded further before transplantation. In combinationwith the uniformity of radial glia marker expression, the data on stabledifferentiation potential for the uncloned LC1 NS cells indicate thatadherent culture in FGF and EGF favours stem cell self-renewal andsuppresses commitment to either glial or neuronal fates.

We found NS cells, obtained by differentiation from ES cells, could betransplanted without formation of tumours over a period of 5 weeks, thusbeing distinct from ES cells which would give rise to macroscopicteratomas within 4 weeks in the mouse brain.

The present invention also relates to nuclear reprogramming methods andto cells and animals obtained via those methods.

Nuclear reprogramming is a technique by which the nucleus of a somaticcell, optionally a stem cell or a terminally differentiated cell, isreprogrammed so as to behave as a nucleus of a cell of relatively higherpotency; ultimately a completely reprogrammed nucleus acts as thenucleus of a pluripotent cell and reprogramming is often taken to meanreprogramming all the way to pluripotency. Methods of reprogramming bynuclear transfer are well established in the art and became wellpublicized after the invention described in WO 96/07732, sometimereferred to as the “Dolly the sheep” invention. Nuclear transfer canthus be used to clone non-human animals.

Nuclear transfer methods have a number of problems. Primarily theyremain of low efficiency, in that the cells obtained are rarelyreprogrammed so as to be truly pluripotent. It is also desirable to beable to carry out genetic manipulation on the nucleus as part of thereprogramming. But, at present this manipulation is not possible or isunreliable due to the difficulty in obtaining clonal populations ofdonor nuclei. Even with clonal nuclei the reprogramming methods arecomplex procedures that require many individual steps. Lastly, ES cellsfrom some species remain difficult to isolate. Using reprogrammingtechniques, if such were available and reliable, may offer analternative route to pluripotent cells in these species.

Further aspects of the present invention have as an object to providealternative approaches to the above problems and to provide solutionsthereto.

Accordingly, the invention provides a method of nuclear reprogramming,wherein the donor nucleus is obtained from a neural stem cell of theinvention.

Neural stem cells of the invention have been found to be reprogrammableat high efficiency, and hence the invention provides an efficientreprogramming method and also facilitates production of geneticallymodified reprogrammed cells, especially pluripotent stem cell, of manyspecies. The ability, via the invention, to propagate individual neuralstem cells clonally means that after genetic manipulation clonalpopulations of cells can be obtained, all with the same geneticmodification, and used in reprogramming methods.

It is also possible to identify neural stem cells according to thepresence of specific cell surface markers and/or the absence ofothers—this has the advantage that the step of culturing according toaspects of the invention described herein can be omitted, as the neuralstem cell can be picked directly from a mixed population, e.g. a brainhomogenate.

A method of nuclear reprogramming is hence provided herein, wherein thedonor nucleus is from a neural stem cell obtained according to anyembodiment or aspect of the invention.

A particular method of nuclear reprogramming, comprises:

obtaining a donor cell;

obtaining a recipient cell;

transferring the nucleus of the donor cell into the recipient cell,wherein the donor cell is (i) a neural stem cell, or (ii) a cellobtained according to a method of the invention; and

culturing the cell so as to reprogram the donor cell nucleus, therebyobtaining a reprogrammed cell.

The nucleus of the recipient cell will generally be removed at one stagein the process so the resulting cell is diploid, this is done optionallyprior to transfer of the donor cell nucleus and optionally aftertransfer of the donor cell nucleus.

One of the interesting possibilities offered by NS cells of theinvention is to introduce genetic lesions e.g. that may inducemalignancy. A further option is thus to genetically manipulate the donorcell nucleus. This can be used to introduce mutations or genes ofinterest, e.g. to generate cells or animals for assays or other testpurposes wherein a plurality of cells are obtained all with the samemanipulation. The range of manipulation is wide. One example is tointroduce a disease-causing genetic sequence or a putativedisease-causing genetic sequence into the donor cell nucleus, useful fordrug screening. A further option is to obtain an animal comprisingtissue derived from the reprogrammed cell and carry out an assay usingthe tissue. It is preferred that the cell is reprogrammed back topluripotency, e.g. by nuclear transfer into an oocyte.

In particular embodiments of the invention, taking advantage of theknowledge from the invention of cell surface makers in the desiredneural stem cells allows omission of cultures steps otherwise needed toisolate a neural stem cell from a mixed population. One suchreprogramming method comprises providing a mixed population of cells,isolating a neural stem cell from the mixed population based upon itscell surface marker profile, and transferring the nucleus of theisolated cell to a recipient cell.

As with the other methods, the isolated cell can be geneticallymanipulated prior to transferring its nucleus to the recipient cell.Also, the isolated cell can be cultured to obtain a clonal population ofcells prior to transfer of the nucleus of one cell in the population tothe recipient cell.

In more detail, an application of the cells of the invention is in highthroughput drug screening. Both neural stem cells of the invention andneurons, glia etc obtained therefrom can be used for the screening, e.g.to identify factors active on either type of cell. The cells or progenycan be used in models of brain cancer. Cells and progeny can be obtainedfrom a tumour of the nervous system, especially the CNS and the neuralcells derived therefrom and their progeny used in screens: the nature ofthe screen is believed to be apparent to all, but in outline comprisesobtaining a cell of the invention or a differentiated progeny thereof,culturing the cell or progeny in the presence of a test factor anddetermining an effect of the factor on the cell or progeny. In aparticular screening use, a cell is provided, which is a cell of theinvention or obtained by the invention and which has been modified so asto express an EGF receptor or an additional EGF receptor.

The cells can be modified before being used in a screen. For example,the cells can be genetically modified to introduce a mutation in a gene,or to introduce a nucleic acid encoding a gene product known to be orsuspected to be implicated in disease, especially a disease of neuralcells including Parkinsonism and Alzheimer's. The Parkin mutation can beintroduced. A gene encoding a protein, e.g. APP, implicated inAlzheimer's can be expressed or mutated or have it expression altered.

Cells of the invention are of use as a source of cells for cell therapy.They offer a source of stem cells from a patient which potentially canbe a source of nuclei for nuclear transfer. They can be used fordelivery of gene therapy, including neuroprotective gene therapy. In anexample therapy, a cell of the invention expresses glial cell derivedneurotrophic factor (GDNF). These cells can be obtained by following theinvention and genetically manipulating the cells. The cells can betransplanted to restore damaged neural circuitry and/or restore brainfunction.

An advantage of the invention lies in the purity of the cells obtained.A pure population is easier to control in transplantation or culture, asin a heterogenous culture a reduced percentage of neurons is obtained asmore cells are already committed to a glia fate. Some therapies willneed both neurons and astrocytes.

Other therapies will need glial cells (e.g. oligodendrocytes for MS,astrocytes, migratory cells, for other applications).

Methods of cloning of non-human animals are provided by the invention.One method of cloning a non-human animal, comprises (i) obtaining aneural stem cell from the non-human animal, (ii) obtaining an oocyte ofthe same species as the non-human animal, (iii) transferring the nucleusof the neural stem cell into the oocyte, and (iv) implanting the cellobtained in (iii) into a female of the same species. The invention henceprovides an efficient method of non-human animal cloning based uponisolation of neural stem cells. The cloning method is believed to be ofapplication to substantially all non-human animals, though especiallydomestic animals including cows, pigs, sheep, cats, dogs, chickens andothers and also laboratory animals including mice and rats.

The invention enables, for the first time, growth of a pure populationof diploid, clonogenic, transfectable, tissue stem cells in the same wayas ES cells. This is a significant step forward in stem cell biologythat opens up a range of new experimental opportunities. For example,previous studies on profiling “neural stem cells” are seriouslycompromised by reliance on heterogeneous neurosphere cultures (e.g.Suslov et al.). Moreover, the radial glia characteristics of NS cellsdefine their in vivo counterpart. The ability to culture and geneticallymanipulate pure populations of radial glia also opens up newopportunities for analysing the cell biology of these remarkable cells,that can function both as stem cells and as specialised scaffold cells.Finally, the ability to propagate NS cells in simple media withoutheterologous cells or cell extracts establishes that self-renewal can bedriven by growth factors alone and does not require a complexmicroenvironmental niche, hitherto considered indispensable by stem cellbiologists. The invention opens up new approaches to nuclearreprogramming and optional genetic manipulation using the cells.

The invention provides efficient generation of neurons from NS cells. Ina method, set out in more detail in a protocol herein, a method ofobtaining neurons comprises (a) culturing neural stem cells in thepresence of an agonist of an FGF receptor and in the absence of anagonist of an EGF receptor; and (b) thereafter, culturing the cells inthe absence of an agonist of an FGF receptor and in the absence of anagonist of an EGF receptor. It is found that the period during whiche.g. EGF is absent primes the cells to become neurons when e.g. FGF issubsequently withdrawn. The neural stem cells are preferably plated inmonolayer culture. Typically, the NS cells are transferred to mediumfree of EGF but which contains FGF-2 and cultured for a period, say atleast 2 days, or at least 4 days, before FGF-2 is also removed from themedium (which includes that cells are transferred to medium that doesnot contain it). In a specific method the cells are grown in FGF-2 butno EGF for a week and then FGF-2 is withdrawn. Withdrawal of FGF leadsto some cell death in the culture, but a good percentage survive andform neurons. This method may be used as an optional addition to any ofthe methods described herein for derivation of NS cells.

It will be appreciated that methods/uses according to all aspects of theinvention can be carried out either in vivo or in vitro.

The methods and compositions of the invention are illustrated in theaccompanying drawings in which:

FIG. 1 shows generation of neural stem (NS) cells from ES cells;

FIG. 2 shows NS cells can be derived from various ES cell line andfoetal forebrain;

FIG. 3 shows NS cells incorporate and differentiate within the adultbrain;

FIG. 4 shows human ES cell or foetal-derived NS cells; and

FIG. 5 shows electrical activity under voltage- and current-clampconditions of NS cells cultured in differentiating medium.

Referring to the figures in more detail, FIG. 1 shows generation ofneural stem (NS) cells from ES cells: A. The adherent NS cell culture(LC1) propagated in EGF and FGF-2 (a) shows no expression of neuronal(b) or astrocyte (c) antigens and uniform expression of the precursormarker RC2 (d) and nestin (not shown). LC1 cells differentiate intoimmunopositive astrocytes (e) on addition of serum and generate neurons(f-h) on growth factor withdrawal. The proportion of neurons obtainedremains >35% of total cells after 115 passages (i). B, Clonal NS-5 cellswere generated through Sox1 neural lineage selection. a and c, Phaseimage of neural precursors at passage 1 and 5 respectively. b and dcorresponding Sox1-GFP fluorescence. e, Single cell 1 hour after platingin Terasaki well. f, Phase contrast image of clonal cell line at passage20. C, RT-PCR for ES cell and neural stem cell/radial glia cell markers(46C, parent ES line; P5, 5 passages after neural differentiation; NS-5clonal NS line; LC1, NS population (passage 17); brain, E12.5/E16.5mouse brain). D, NS-5 immunoreactivity for neural stem cell/radial gliamarkers. E, NS-5 differentiation into astrocytes (a,b) and neurons (d,e)with loss of nestin immunoreactivity (c, f) F, Colonies of NS-5 cells(a) generate neurons on growth factor withdrawal (b) and in the presenceof EGF/FGF retain homogenous expression of RC2 and BLBP with noimmunoreactivity for GFAP (c,d). G, Metaphase spread of NS-5 (passage31).

FIG. 2 shows NS cells can be derived from various ES cell line andfoetal forebrain: NS cells were derived from independent ES cell lines(CGR8, E14Tg2a) or primary cortical (Cor-1) and striatal (Str-1) tissue.A, RT-PCR of stem cell/radial glia markers. B, RT-PCR fortranscriptional regulators. C, The ES-derived line (CGR8-NS) andfoetal-derived line (Cor-1) are indistinguishable from LC1 by morphology(a, f) and neural stem cell/radial glial marker immunoreactivity (b, c,g, h), and can each differentiate into neurons (d, i) and astrocytes (e,j).

FIG. 3 shows NS cells incorporate and differentiate within the adultbrain: a-h, Confocal images of LC1 NS cells, lentivirally transducedwith eGFP, four weeks post-grafting into hippocampus (a, b) or striatum(c-h). b, d, higher magnification of the insets in panels a and c,respectively. e, f, Examples of eGFP grafted NS cells (green) showingco-expression (yellow) of the neuronal markers TuJ (e, red) or MAP-2 (f,red). g, astroglial marker GFAP (red). h, neural progenitor markernestin (red). i, Quantitative analysis of graft-derived neuronal (MAP2),astroglial (GFAP), progenitor (Nestin), and proliferating (Ki67) cells,four weeks after transplantation into adult mouse striatum. Data aremeans (±standard deviation) of at least 500 eGFP+ cells from fiveindependent animals. Scale bars: a,c, 100 □m; b, d ,e, 40 □m.; f-h, 20□m.

FIG. 4 shows human ES cell or foetal-derived NS cells: A, Derivationfrom human ES cells. a, human ES primary culture. b, differentiation ofhuman ES cells into neural-rosette structures. c, passage 9 in NSexpansion medium. d, individual cells exhibit radial glial morphology.e-h, immunostaining for neural stem cell/radial glia markers. B,Derivation from human feotal forebrain. i, neurospheres generated fromcortex. j, attachment and outgrowth. k, passage 5 in NS expansionmedium. I, radial glia morphology. m-p, neural stem cell/radial glialmarkers. C, Differentiation. q, TuJ positive immature neuron. r, GFAPpositive astroglia.

FIG. 5 shows: A. Superimposed inward and outward current tracingsobtained at different membrane potentials (between −70 and +40 mV from aholding potential of −90 mV), from three different NS cells followingincubation in differentiating medium for six (a), twenty (b) and thirtydays (c). B. superimposed voltage responses obtained following injectionof depolarising rectangular current pulses in the same three cells (a, band c) as in (A) by switching from voltage- to current-clamp immediatelyafter current recordings shown in (A) were obtained. The dashed linerepresents a voltage level of −60 mV. C. Average Na⁺ currents elicitedat −20 mV from cells cultured in differentiating medium for increasingtimes as indicated by labels. Bars indicate SE. D. Superimposed inwardcurrents elicited at −40 mV and 0 mV in 10 mM Ba²⁺ and in the presenceof TTX; the holding potential was −90 mV. E. Current/voltagerelationship from the same cell as in (D).

The invention is demonstrated in use by the following examples.

EXAMPLES Example 1-1

Isolation and Culture of Bulk Populations of NS Cells

An ES cell differentiation protocol was recently established that allowsefficient and consistent generation of between 50-70% Sox1 positiveneural precursors in adherent monolayer culture. In order to isolate,expand, and characterise the neural precursors (derived from mice)within these cultures, a previously generated cell line (46C cells) wasused which contained a GFP-IRES-puromycin reporter cassette targeted tothe Sox1 locus (Ying et al., 2003b). Puromycin was added to thedifferentiation cultures after 7 days, and within 3 days more than 95%of remaining cells were GFP+ neural precursors. At this point, cellswere re-plated in N2B27 media supplemented with EGF/FGF-2 (withoutpuromycin). These cells grew rapidly, within a few passages taking on ahomogeneous morphology.

These 46C neural precursor cells (46C-NP) were kept continuously inculture for more than 20 passages retaining a characteristic bipolarmorphology. These cultures showed very little cell death and had a highplating efficiency with doubling time of approximately 25 hrs.

Example 1-2

Culture of Cell Lines

To isolate clonal cell lines, single cells from passage 5 bulk culturesof 46C-NP cells were plated into separate wells of a microwell plate. Of95 single cells scored, 15 were grown up into colonies and, of these, 4lines were kept growing continuously through more than 10 passages. Oneline (NP5) showed the characteristic bipolar cells with homogenousmorphology identical to that observed in the bulk population. This NP5line has been maintained in culture indefinitely (NP5 passage 41; morethan 5 months).

Example 1-3

Characterization of NS Cells

In order to characterise both the bulk cell population and clonal linesimmunocytochemistry was used for a range of markers. As expected, eachof the cell lines was found to be positive for the neural precursormarkers, nestin or vimentin. Importantly, these cells were also positivefor the radial glia specific markers RC2, 3CB2 and astrocyte-specificglutamate transporter (GLAST). Less than 1% of cells were positive foreither GFAP or b III tubulin suggesting that very little spontaneousdifferentiation to astrocytes or neurons occurs during passage of thesecells. Unlike primate/human cells, rodent radial glia are negative forGFAP. Furthermore, these cells are immunoreactive for SSEA-1, anantibody that recognises the LewisX antigen, previously used to enrichfor adult neural stem cells.

RT-PCR was performed with a range of markers to confirm their identityas radial glia. All four clonal lines as well as the bulk populationwere negative for Oct4 or Nanog confirming that they were not ES cells,while they did express nestin, vimentin and GLAST, consistent with theimmunocytochemistry. Furthermore, each cell line showed expression ofBLBP, a radial glia marker. These cells also express Musashi-1 andprominin.

Interestingly, the bulk culture of 46C-NP gradually lost expression ofSox1, as assessed by the GFP reporter, such that by passage 5 (˜2 weeks)very few cells remained green. Also, none of the clonal lines showedSox1 driven GFP expression. RT-PCR confirmed that these cells did notexpress Sox1, but instead expressed the related SoxB1 class protein,Sox2. Taken together these results suggest that a range of Sox1expressing neural precursors can be isolated using a puromycin selectionstrategy, and that Sox1−, Sox2+ cells with properties of forebrainradial glia can be isolated and clonally expanded.

Example 1-4

NS Cell Line Differentiation into Neurons and Glia

The molecular markers expressed by the NS cell lines confirm that theyare a type of neural precursor cells. To confirm that these cells weretrue multipotent stem cells (i.e. capable of differentiation as eitherneurons or glia) a range of conditions designed to test the potency ofthe cells were tested.

Previous investigations of neural precursors induced differentiationthrough protocols involving withdrawal of mitogens and/or addition ofserum or other cytokines. In the present case, EGF, FGF, or both, waswithdrawn from proliferating NP5 and 46C-NP cells cultured on plastic.Simultaneous removal of both EGF and FGF resulted in rapid and extensivecell death within 24 hrs. Culture with FGF alone, by contrast, lead tocell death over the course of 3 days. Culture in EGF alone resulted inno cell death or differentiation, but cell proliferation was reduced.Thus, EGF clearly acts an essential cell survival signal (partiallycompensated by FGF signalling) for NS cells cultured on plastic.

The effect of serum on proliferating cells after withdrawal of cytokineswas also tested. Cells treated in this way survived, even in the absenceof EGF, and rapidly differentiated in a synchronous manner such that100% of cells acquired a large flat astrocyte that stained positive forGFAP and negative for nestin. Therefore, all NS cells within aproliferating population are capable of differentiation as astrocytes.This effect can be mimicked by addition of BMP4 in absence of serumfollowing EGF/FGF withdrawal on plastic. CNTF and TGF-b behavedsimilarly to BMP4, although the astrocyte morphology was different andthere was more cell death initially. BMP, CNTF and LIF have each beenshown to induce a astrocyte fate from primary cortical progenitors(Gross et al., 1996; Lillien and Raff, 1990; Nakashima et al., 1999).Consistent with these studies, a rapid induction of Id genes (>30 fold)with BMP-4 or serum treatment was found (data not shown).

In an attempt to induce neuronal differentiation, the withdrawal of EGFon laminin-treated dishes was tested. To avoid cell death seen withculture of cells on plastic, the cells were plated on a lamininsubstrate in the presence of FGF, but no EGF. The PCD seen in theseconditions on plastic or gelatin did not occur and cells survived. Therewas a change in morphology of the cells to more extended bipolarprocesses, with characteristic endfeet of radial glia. These cellsshowed a drastic reduction in proliferation (through BrdU incorporation)but maintained radial glia markers (RC2, vimentin, nestin) and did notdifferentiate, as only a small proportion of neurons or astrocytes wasseen in regions of high density. To induce neuronal differentiation,after 6 days the media was changed from NSA/N2 +FGF2 to NSA/B27 withoutFGF2. This promoted around 40-60% neuronal differentiation of cells asjudged by MAP2 and bill tubulin antibody staining. These neurons wereGABAergic as assessed by GABA immunoreactivity, while there were fewastrocytes (GFAP). These results are consistent with the previouslydescribed role of FGF in preventing neuronal differentiation.

It was also found that each cell line could be frozen and thawed withsimilar success to ES cells.

In further tests, the differentiations were attempted starting fromsingle cells. This clonal expansion and differentiation showed all thecells to have ability to form neurons.

Example 1-5

Isolation of NS Cells from any ES Cell Line Without Use of GeneticSelection Strategies

The N2B27 media used to isolate NP5 was originally optimised forconversion of ES cells to neural precursors and hence allowed goodsurvival of both ES cells and neural progenitors. Thus, in the absenceof puromycin selection, it was not possible to efficiently expand 46C-NPcells with EGF and FGF-2 due to carry-over of ES cells and non-neuralcell type (data not shown). To overcome this and allow the generation ofradial glia cells from other non-targeted ES lines, other basal mediacombinations were tested that allowed survival and expansion of neuralprecursors but not other cell types.

Using a commercially available media, NS-A media (Euroclone), it wasfound that re-plating of a 46C ES cell monolayer differentiation at day7 resulted in formation of clumps/spheres of cells, as well as a celldeath of non-neural cells. Subsequent attachment of these cell clumpsoccurred and a homogeneous population of cells were outgrown. Furthercharacterization of this passagable population (>75 passages) of cellsrevealed an identical profile of expressed markers seen previously usingthe puromycin selection strategy. Thus, the cells were positive forneural precursor markers (nestin, vimentin) and also radial glia markers(RC2, GLAST, BLBP and Pax6). 46C-NP cells behaved similarly in eitherN2B27 or NSA media once established with only small differences inmorphology, and no differences in marker genes.

Using this protocol, cell lines from six other ES lines were isolated:CGR8, E14T, Oct4-GIP, S11, R1 and V6.5. Each of these cell lines has asimilar morphology and expression profile and can be passagedextensively.

Example 1-6

Transplantation of NS Cells

To test the potential of the NS cells in vivo, they were transplantedinto both embryonic, and adult CNS, as well as in kidney capsule graft.

Electroporation was tested on NP5. They were found to be electroporatedefficiently using a square wave elecroporator. The cells are alsotransfectable using lipofectamine plus reagent. This is a majoradvantage as all genetic manipulations used in mouse now becomeavailable.

To allow rapid evaluation of the grafted cells, 46C-NP were transducedwith lentiviral particles carrying the eGFP marker gene. The infectionwas highly efficient and almost 95% of the cells were successfullymarked, the eGFP signal being strong and stable with passages. The useof the lentiviral infection allowed the transgene to be stablyintegrated and the signal to remain stable for long term analyses of thegrafted cells.

The behaviour of the cells following transplantation in the embryonicbrain (an environment that contains all the molecules and factors ableto sustain and direct differentiation of immature neural cells) wasevaluated. 100,000 cells were resuspended in a final volume of 2microlitres following the procedure previously described by Magrassi andcolleague (Magrassi et al., Development 1999) using E14.5 mouse embryosas recipients. The cells survived well after transplantation (anapproximative evaluation of around 20000 cells in the graft was made),the eGFP signal was easily detectable and they displayed migrationalactivity already at early time points after the grafting. The fates thatthe grafted cells acquired at different time points (4 days, 7 days, 2weeks and 1 month) after the grafting were analysed. At the 4 day and 1week time points, the majority of the cells were still immature asindicated by the nestin immunoreactivity, while 23% expressed theneuronal marker Tuj-1 and 16.3% acquired the glial marker GFAP. Thesedata indicate that the transplanted NS are able to generate both neuronsand glia in vivo as expected from multipotent NS cells.

The NS cells were also transplanted into the adult striatum. In thesetransplants, the cells survived well (the survival is anyway lower thanin the embryonic grafts) and differentiated toward both neuronal andglial fates. Quantitative analyses performed at 2 weeks aftertransplantation indicated that 43.3% of the cells expressed theneuronal-specific marker Tuj-1, while 26.6% displayedimmunoreactivityforthe glial marker GFAP. A small fraction of the cells(11.1%) retained an immature phenotype as indicated by the expression ofnestin.

NP5-GFP cells into kidney capsule did not give rise to teratomas (n=4,data not shown).

Example 2 Example 2-1

Methods

Mouse Cell Culture and Differentiation

ES cells and neural differentiation are detailed by Ying & Smith, 2003.NS cells termed LC1 and other ES cell-derived NS cells were routinelygenerated by re-plating day 7 neural differentiation monolayer cultureson uncoated plastic in NS-A medium (Euroclone) supplemented with N2 and10 ng/ml of both EGF and FGF-2 (NS expansion medium). Cells formedaggregates which subsequently attached and outgrew NS cells. Cellstermed 46C-NS cells were generated after addition of 0.5 μg/ml ofpuromycin to differentiating adherent cultures at day 7. Cells werere-plated 3 days later into an uncoated T75 flask in N2B27 media with 10ng/ml of both EGF and FGF-2 (Peprotech) in the absence of puromycin. Aclonal line, NS-5, was generated by plating single cells into 96-wellmicrowell plates (Nunc) by limiting dilution (single cells scored onehour after plating). Primary cultures were generated using standardprotocols from cortex/striatum of E16.5 mouse embryos and allowed toattach on flasks treated with 0.1% gelatin. Cor-1 and Str-1 cells werethen expanded on gelatin using NS expansion medium. For astrocytedifferentiations, NS cells were replated onto 4-well plates at 1×10⁵cells/well in NS-A medium supplemented with 1% fetal calf serum or 10ng/ml BMP4 (R&D System). For neuronal differentiation 5×10⁴ NS cellswere plated into poly-ornithine/laminin treated wells in NS-Asupplemented with FGF-2 alone. After 7 days the media was switched toNS-A supplemented with B27 (Gibco) without growth factor. For clonaldifferentiation, 1000 cells from NS-5 or Cor-1, cultures were plated in10 cm plates pre-treated with laminin, expanded for 12 days inEGF/FGF-2, and differentiated in situ as above.

Characterization of NS Cells

Immunocytochemistry was performed using appropriate TRITC or FITCsecondary conjugates and nuclear counterstaining with DAPI. Primaryantibodies were used at the following dilutions: Nestin (1:10), Vimentin(1:50), Pax6 (1:5), 3CB2 (1:20), RC2 (1:50) (DSHB); TuJ (1:200)(Covance); GFAP (1:300) (poly and mono, Sigma); MAP2 (1:200) (Chemiconand Becton Dickinson); NeuN (1:200), GABA (1:200), Gad65/67 (1:200)(Chemicon); Synaptophysin (1:200) (Sigma); Olig2 (1:5000) (H.Takebayashi); Emx2 (1:2000) (A. Corte); BLBP (1:500) (N. Heintz);prominin/mAb13A4 (1:200) (W. Huttner). Negative controls were ES cells,differentiated NS cells or secondary alone. For RT-PCR, total RNA wasextracted using RNeasy kit (Qiagen), and cDNA generated usingSuperscript II (Invitrogen). PCR was performed for 30 cycles for allmarkers except β-actin (25 cycles). For metaphase spreads cells weretreated with 5 mls of 0.56% KCl for 20 mins, fixed in methanol:aceticacid (3:1) on ice for 15 mins, spread onto glass slides and stained withTOPRO-3 (Molecular probes).

Human Embryo and Foetal Cultures

Research on human tissue with informed consent was approved by theResearch Ethics Committee of Lothian Health Board. Frozen supernumeraryhuman embryos were donated for research under licence R0132 issued bythe Human Fertilisation and Embryology Authority. Human cortex wasdissected from a Carnegie stage 19/20 foetus following electivetermination with consent for research under the Polkinghorne guidelines.

Transplants

Foetal surgery was performed as described by Magrassi et al, 1998. Usinga glass capillary, 5×10⁴ cells in a volume of 1 μl of HBSS were injectedinto the telencephalic vesicles of E14.5 Sprague Dawley rat foetusesexposed under transillumination. Injected foetuses were replaced intothe abdominal cavity for development to term. After delivery animalswere sacrificed at seven days (Postnatal day (P) 1, n=16) and five weeks(P30, n=8) post transplantation. For adult transplantations, 129 or CD1mice were placed in a Kopf stereotaxic frame and received an injectionof 2×10⁵ NS cells suspended in 5 μl of HBSS into the striatum (n=22) orhippocampus (n=21). Transplanted mice were sacrificed after two (n=16)and four weeks (n=10) and perfused transcardially with 4%paraformaldehyde. Cryosections (16 μm) were stained with the followingantibodies: (mouse): NeuN (1:100) and Ki67 (1:10) (Chemicon), MAP2(1:200; Becton Dickinson), Nestin (1:5; Ron McKay); (rabbit): βIIItubulin (1:500; Covance); GFAP (1:200; Dako); secondary antibodies,Texas Red (Vector) (Jackson ImmunoResearch) and AlexaFluor 488(Molecular Probes). Sections were preserved in antifading solution andanalysed on Nikon TE2000-S ECLIPSE and Biorad Radiance 2100 confocalmicroscopes.

Example 2-2

We induced monolayer differentiation of ES cells for 7 days thenreplated in basal medium (NS-A plus N2) supplemented with EGF and FGF-2.Cells surviving under these minimal conditions (non-permissive for EScell survival) predominantly associate into floating clusters. After 3-5days these aggregates were harvested and replated in fresh medium. Theyattached within 2-3 days and outgrew a morphologically homogeneouspopulation of bipolar cells, named LC1. Upon passaging LC1 cellscontinued to grow as adherent cultures, often forming lattice networks.They can be continuously and rapidly propagated with a doubling time ofapproximately 24 hours. LC1 cells display the neural precursor markersnestin and RC2 but expression of the astrocyte differentiation markerGFAP or of neuronal antigens is negligible (FIG. 1A). On exposure toserum or BMP, LC1 cells adopt typical astrocyte morphology within 48hours and subsequently uniformly express GFAP (FIG. 1A,e). In contrast,cells with neuronal processes appear after replating on laminin withoutEGF for 5-7 days and then withdrawing FGF-2. These cells expressneuronal markers type III β-tubulin, MAP2 (FIG. 1A, f,g) and neuN (notshown). Most neurons stain for GAD67 (FIG. 1A,g) and GABA (not shown)and by 7 days a sub-population show expression of the mature markersynaptophysin (FIG. 1A, h). High numbers of neurons (>35%) are generatedeven after 115 passages (FIG. 1A,i). Together with the observation thatLC1 cells retain a diploid chromosome content at late passages (notshown), this confirms the presence of self-renewing neural stem (NS)cells.

Example 2-3

To determine whether cell aggregation is essential for generation of NScells, we maintained cell attachment throughout the derivation process.For this we exploited lineage selection (Li et al, 1998) using 46C EScells in which the GFPirespac reporter/selection cassette is integratedinto the Sox1 gene, a specific marker of neural specification (Aubert etal, 2003). Transient puromycin selection after differentiation inductionyielded a purified population of neural precursors without residual EScells (Stavridis et al, 2003) (FIG. 1B a,b). FGF-2 and EGF were thenapplied to the Sox1 expressing neural precursors in enriched medium.Cells remained adherent in this condition. Cellular heterogeneityreduced over 3-4 passages as bipolar cells progressively increased innumber and began to form extensive lattices. Intriguingly, expression ofSox1 was lost at this stage (FIG. 1B c,d) but the cells remainedpositive for Sox2 and nestin. To establish the presence of neural stem(NS) cells, single cells were isolated in Terasaki wells and expanded asadherent cultures (FIG. 1B, e,f). Initially 5 clonal lines were derivedof similar morphology and growth characteristics to the bulk LC1population. These cells lacked detectable expression of the pluripotencyfactors Oct4 and Nanog, and also of the early neural marker Sox1, butretained the pan-neuroepithelial markers Sox2 and nestin (FIG. 1C). NScells were thus generated through a Sox1 positive early neuroectodermalprecursor via continuous adherent culture.

Example 2-4

Clone NS-5 was examined in further detail and found to express Pax6,Glast, and BLBP mRNAs, and to be immunopositive for RC2, vimentin, 3CB2,SSEA1/Lex1 and prominin (FIG. 1D). This set of markers is considereddiagnostic for neurogenic radial glia, the precursors of both neuronsand astrocytes during development of the nervous system (Campbell et al,2002; Hartfuss et al, 2001). As with uncloned LC1 cells, NS-5 cells werecompetent for astrocyte and neuronal differentiation (FIG. 1E). NS-5cells plated at clonal density in EGF plus FGF-2 gave rise to coloniesof bipolar cells. Every colony showed expression of RC2 and BLBP invirtually all cells and absence of GFAP (FIG. 1F, c,d). These subclonescould be picked and continuously expanded. To determine the frequency ofcells within NS cultures that are capable of neuronal differentiation weagain plated NS-5 cells at clonal density, expanded for 12 days inEGF/FGF-2 followed by FGF-2 alone for 5 days, then a further 7 dayswithout growth factor. Every colony (126/126) produced TuJ positivecells (FIG. 1F, b). These data indicated that all colony forming cellsin NS cultures were competent for neuronal differentiation. Finally,like LC1, NS-5 cells maintained a diploid chromosome complement (FIG.1G) and represented a non-transformed clonal neural stem (NS) cell linethat self-renewed without a requirement for a complex cellularmicroenvironment.

Example 2-5

To assess whether the serum-free monolayer induction protocol is aprerequisite for NS cell generation, ES cells were induced todifferentiate by embryoid body formation and exposure to retinoic acidin serum-containing medium (Bain et al, 1995). Aggregates were subjectedto Sox2 lineage selection (Li et al, 1998; Billon et al, 2002) with G418for 48 hours then replated in the presence of FGF-2 and EGF withoutserum. Following attachment, Sox2 positive, nestin positive, cellsproliferated that displayed the bipolar morphology and lattice growthtypical of NS cells plus capacity for astrocyte and neuronaldifferentiation after multiple passages (data not shown). Thus NS cellswere derived from embryoid bodies.

Example 2-6

NS cells were derived using monolayer induction without lineageselection as described for LC1 from three independent ES cell isolates,E14TG2a, CGR8 and R1. All NS lines examined expressed nestin and RC2 inat least 95% of cells. More detailed inspection of CGR8 and E14TG2aderived NS cells showed the full panel of radial glia markers (FIG. 2A),the neural precursor markers Sox2 and Sox3, plus the bHLH transcriptionfactors Olig2 and Mash1 (FIG. 2B). Down-regulation of Sox1 butmaintenance of Sox2 was a striking feature of these NS cells, in view ofthe postulated determinative function of these transcription factors(Pevny et al, 1998). Thus whilst Sox1 marks all neuroectodermalprecursors, it was not retained in stem cells where Sox2 is likely toplay a key role. The NS cells also expressed Emx2 which is implicated inexpansion of neural precursor cells (Hein et al, 2001; Galli et al,2002). All NS cultures tested underwent astrocyte and neuronaldifferentiation assessed by both morphology and immunostaining (FIG.2C).

Example 2-7

In light of their apparent relationship to radial glia, we examinedwhether NS cells resulted from the pre-adaptation of ES cells to ex vivopropagation or could be derived from foetal neural tissue. Primaryfoetal CNS cells from E16.5 foetal brain adhered poorly to plastic inbasal medium plus growth factors and spontaneously formed aggregates.After 6-7 days these aggregates settled onto gelatin-coated plastic.Fourteen days later, outgrowths were trypsinised and plated ontogelatin-coated plastic. In three separate experiments cellsmorphologically identifiable as NS cells proliferated and weresubsequently expanded into continuous cell lines. These foetal brainderivatives expressed the same radial glia and neurogenic markers as theES cell derived NS cells (FIG. 2A,B ) and showed consistent mRNAprofiles. They were likewise competent for astrocyte and neuronaldifferentiation (FIG. 2C). Cortex-derived Cor-1 cells were plated assingle cells then colonies subjected to sequential growth factorwithdrawal as described for NS-5. Every colony produced TuJ positiveneurons. This indicated that all clonogenic cells in the Corn culturewere neurogenic. Cor-1 cells were also readily sub-cloned andcontinuously expanded from individual cells, indicative of self-renewal.Thus NS cells were derived from foetal brain and shared the keyproperties of ES cell derived NS cells.

Most NS cells had the elongated bipolar morphology, lamellateextensions, end-feet and oval nuclei anticipated for radial glia (Rakic,2003). Flattened and compact cells with short extensions were alsopresent. Immunostaining for the metaphase marker phosphorylated histoneH3 indicated that the compact cells are mitotic. Time lapsevideomicroscopy confirmed the dynamic change in morphology prior to celldivision. In addition, the time lapse revealed that NS cell undergointerkinetic nuclear migration, a well-characterized feature ofneuroepithelial and radial glia cells in vivo.

The NS cells were hence continuously expandable in vitro analogues ofneurogenic radial glia.

Example 2-8

Frozen/thawed passage 40 mouse neurospheres were allowed to attach togelatin-coated plastic in NS expansion medium. Bipolar cells outgrewthat are indistinguishable from NS cells. These cells can be seriallypropagated as uniformly RC2 positive, GFAP negative, populations andthen induced to differentiate into astrocytes or neurons.

Example 2-9

We investigated the behavior of NS cells upon transplantation into mousebrain. ES cell derived LC1 cells transduced with a lentiviral eGFPexpression vector were introduced into the developing brain byintra-uterine injection at E14.5 (Magrassi et al, 1998). Animals weresacrificed after birth and the presence of eGFP positive cells examinedin brain sections. NS cell progeny had migrated into various brainregions. Immunohistochemical analyses revealed co-expression of eGFPwith the precursor marker nestin, neuronal markers TuJ, NeuN and MAP2,and in lesser numbers with GFAP. NS cells were also injected into theadult mouse striatum. In this case GFP-positive cells remained localisedto the vicinity of the injection site. Four weeks after grafting,44.4±5.7% of GFP expressing cells had neuronal morphology and wereimmunopositive for MAP2, 37.4±6.1% expressed GFAP, and 4.2±1.9% retainedexpression of nestin (FIG. 3). The proliferative marker Ki67 wasdetected in only 1.0±0.6% of GFP positive cells indicating that NS cellswithdraw from the cell cycle in vivo. Consistent with this we observedno histological evidence of un-regulated proliferation or tumourformation in a total of 35 brains examined one month aftertransplantation. Furthermore, NS cells grafted to mouse kidney capsulesdid not proliferate or give rise to teratomas. These data indicated thatNS cells can survive and differentiate in both foetal and adult brainenvironments and unlike ES cells (Brustle et al, 1997), they do not giverise to teratomas. Moreover, the relatively high frequency of neuronaldifferentiation is in marked contrast to grafts of passaged neurospheres(Rossi et al, 2002).

Example 2-10

Finally we investigated whether similar NS cells could be isolated fromhuman sources. In the process of deriving human ES cells from donatedsupernumerary embryos, we observed after 5-6 weeks of culture extensivedifferentiation into rosette structures typical of neuroepithelialcells. These cells were transferred into NS expansion medium. After afurther 3-4 weeks bipolar cells similar to NS cells emerged from thesecultures (FIG. 4A) and have been continuously cultured for 5 months. Wealso sourced Carnegie stage 19-20 human foetal cortex tissue from anelective termination. Dissociated cells initially formed floatingaggregates that after 7 days were replated and allowed to attach togelatin-coated plastic as for derivation of NS cells from mouse foetalbrain. A proliferating culture was established (FIG. 4B). Human NScultures from both ES cells and fetal tissue were characterized by thepresence of flattened cells associated with the bipolar cells. However,all cells expressed immature precursor markers nestin, vimentin and 3CB2(FIG. 4B). Time lapse monitoring confirmed that the two cellmorphologies are plastic and interconvertible. These human cells alsoexhibited low levels of GFAP consistent with the activity of the humanGFAP promoter in radial glia (Rakic, 2003; Malatesta et al, 2000). Theyproliferated more slowly than the mouse cells, with doubling times ofseveral days. After sequential withdrawal of EGF and FGF-2 they appearedto generate immature neuronal cells (FIG. 4C). Cells of typicalastrocyte morphology with intense GFAP immunoreactivity were producedafter passaging in serum (FIG. 4C).

Example 3

The electrophysiological properties of NS cells in culture wereinvestigated during in vitro differentiation by employing the whole-cellvariant of the patch-clamp technique, in order to know whether,following specific treatments, these cells could be efficientlytransformed into mature and functional neurons from anelectrophysiological point of view.

Solutions for Electrophysiological Recording

Seals between electrodes and cells were established in a bath solutionconsisting of (in mmoles/l): 155 NaCl, 1.0 CaCl₂, 1 MgCl₂, 3.0 KCl, 10glucose, 10 HEPES/NaOH (pH 7.4). After establishing the whole-cellconfiguration, for current-clamp recording and for total currentrecording in voltage-clamp, the pipette filling solution contained (inmmoles/l): 128 KCl, 10 NaCl, 11 EGTA, 4 Mg-ATP, 10 HEPES/KOH (pH 7.4).For the study of voltage-gated Na⁺ channels under voltage clampconditions, the patch pipette was filled with (in mmoles/l): 130 CsCl,10 NaCl, 20 TEA-Cl, 10 EGTA, 2 MgCl₂, 4 Mg-ATP, 10 HEPES/CsOH (pH 7.4)and the extracellular solution contained (in mmoles/l): 130 NaCl, 2CaCl₂, 2 MgCl₂, 10 glucose, 5 tetrethylammonium-Cl, CdCl₂ 0.2, 10HEPES/NaOH (pH 7.4). For the study of voltage-gated Ca²⁺ channels, thepatch pipette was filled with (in mmoles/l): 120 CsCl, 20 TEA-Cl, 10EGTA, 2 MgCl₂, 4 Mg-ATP, 10 HEPES/CsOH (pH 7.4) and the extracellularsolution contained (in mmoles/l): 130 NaCl, 10 BaCl₂, 10 glucose, 5tetrethylammonium-Cl, 10 4-AP 1, TTX 10⁻³, HEPES/NaOH (pH 7.4).

Patch Clamp Recording

Ionic currents were recorded under voltage-clamp conditions using thepatch-clamp whole-cell configuration (19) at room temperature (20-24°C.) with an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Burlingame, Calif.) and digitized at sampling intervals of 26-100 μsecusing a Digidata 1322A A/D converter (Axon Instruments Inc., Burlingame,Calif.) interfaced with an IBM-compatible PC. Stimulation, acquisition,and data analysis were carried out using the following softwarepachages: pClamp 9 (Axon-instruments Inc., Burlingame, Calif.) andORIGIN 6 (Microcal Software Inc., Northampton, Mass.). For voltage-clampexperiments linear components of leak and capacitive currents were firstreduced by analogue circuitry and then almost completely canceled withthe P/N method. Patch pipettes were made from borosilicate glass tubingand fire polished. Pipettes had a final resistance of 3-4 MΩ when filledwith internal solution. Currents were filtered at 5 KHz.

FIG. 5A shows current recordings obtained during whole-cellvoltage-clamp steps to depolarising test potentials in three NS cells atdifferent stages of in vitro differentiation (cultured respectively for6, 20 and 30 days in a differentiating medium), using bath and pipettefilling solutions suitable for the isolation of both inward (Na⁺) andoutward (K⁺) voltage-gated ionic currents. From a simple inspection ofthe current tracings it is evident that a sizeable outward voltage-gatedcurrent is already present at early stages of in vitro differentiation(6 days, trace a). This current, blocked by application of anextracellular saline containing 5 mM tetraethylammonium-Cl, has thefeatures of a delayed-rectifier K⁺ current. Its amplitude increased onlyslightly at later stages of differentiation (20-30 days, traces b and crespectively). By contrast, the amplitude of the inward current,negligible after six days, increases dramatically by increasing the timeof exposure to the differentiating medium. FIG. 5B shows the voltageresponses elicited following intracellular injection of rectangulardepolarizing current stimuli between 70 and 300 pA from a holdingpotential of about −80 mV, after switching from voltage-clamp tocurrent-clamp mode in the same cells shown in FIG. 5A. Indeed, theincrease in amplitude of the voltage-gated inward current reflects thecapacity of the differentiating cells to elicit action potentials. Infact, the cell exposed for only six days to the differentiating agentsand showing a negligible inward current was not able to elicit aregenerative voltage response (trace labelled with (a) in FIG. 5B). Bycontrast, an overshooting action potential with a relatively fastdepolarisation rate could be elicited in the cell cultured indifferentiating medium for thirty days (trace labelled with (c) in FIG.5B) and displaying a large inward current (trace c in fig. A). Anintermediate situation, with an abortive action potential, was found inthe cell treated for twenty days with the differentiating agents (tracelabelled with (b) in FIG. 5B) and displaying a moderate inward current(trace b in fig. A). From this preliminary analysis it turns out thatthe excitability properties of these cells are strictly correlated tothe magnitude of the inward voltage-gated conductance. For aquantitative analysis of this conductance as a function of the time ofexposure to the differentiating medium, the inward currents wereelicited using cells at different stages of differentiation and applyingintra- and extracellular salines specific for the study of the activityof voltage-gated Na⁺ channels (see methods). At any time of celldifferentiation the fast inactivating inward current was completelyblocked by the selective Na channel blocker tetrodotoxin (1 □M) andpeaked at a test potential between −20 and −10 mV, showing the typicalfeatures of voltage-gated Na⁺ currents in neurons (data not shown). InFIG. 5C the development of the Na⁺ current amplitude at −20 mV as afunction of the time of exposure to the differentiating medium is shown.Interestingly, the increase of the Na⁺ conductance well correlates withthe increasing cell exposure to the differentiating medium. Indeed, onaverage the Na⁺ current amplitude increases of about a factor ten within30 days of treatment (from −55±14 pA (n=17) during the first 5 days to−434±135 pA (n=13) for more than 25 days). In the same way, theregenerative potential (the □V measured between the threshold and thepeak) elicited by the Na⁺ current under current clamp conditions rangedbetween 0 and +20 mV during the first 15 days of in vitrodifferentiation (n=6), whereas following more than 25 days of treatmentit reached values between +30 and +70 mV (n=6). On the whole, theexcitability properties and those of the underlying voltage-gated Na⁺conductance found in NS cells treated longer than 25 days with thedifferentiating medium are typical of neurons developing toward theiradult phenotype. Another feature corroborating the previous conclusionsis the presence of voltage-gated Ca²⁺ channel conductances in most ofthe tested cells following exposure to the differentiating medium for atleast seven days or longer. FIG. 5D shows sample tracings obtained bymembrane depolarization to the indicated test potentials from the samecell bathed in 10 mM Ba²⁺. The fast activating and relatively fastinactivating (□_(h)=21 ms) current component elicited at −40 mV isreminiscent of the neuronal LVA Ca²⁺ channel current (Carbone and Lux,1987). By contrast, the Ba²⁺ current elicited at 0 mV, displaying a slow(□_(h)=73 ms) and incomplete inactivation, has the typical features ofthe neuronal HVA Ca²⁺ channel current. The presence in this cell of twodistinct, LVA and HVA, Ca²⁺ channel conductances is confirmed by thecurrent-voltage relationship of FIG. 5E. On average the LVA currentpeaked at −40 mV, while the I/V relationship for the HVA current peakedat 0 mV. A HVA Ba²⁺ current was detectable in 19 out of 27 cells, whilea LVA current component was measured in 60% of the cells alreadyexpressing a HVA Ca²⁺ current (n=13).

This data shows that neurons produced from neural stem cells of theinvention can function: they can fire action potentials, the hallmark ofactive neurons.

Thus, according to the invention, we have derived and homogenouslypropagated defined NS cells as differentiated progeny of ES cells andfoetal brain extracts. The NS cells proliferated clonally in simpleadherent monoculture and remained diploid. After prolonged expansion,they differentiated efficiently into neurons and astrocytes both invitro and on transplantation into the adult brain. NS cells also formedoligodendrocytes in vitro. NS cells uniformly expressed morphologicaland molecular features of radial glia, foetal precursors of neurons andastrocytes. We were able to establish adherent NS cell lines from mouseand human foetal brain.

Example 6

NS cells were obtained from mouse ES cell and foetal cortex using theprotocol below. The derived NS cells uniformly expressed radial gliamarkers. NS cells derived specifically from CGR8 ES cells or from E16foetal cortex (internal reference: Cor-1 and clonal derivative Cor-1.3)were analysed for expression of the indicated markers byimmunochemistry. Examination at high power showed that the radial gliamarkers were each expressed in almost all cells whilst they wereuniformly negative for GFAP.

Example 7

NS cells were derived from expanded mouse foetal forebrain neurospheresusing the protocol below. An NS line derived from a long-term foetalneurosphere culture (40 passages) exhibited identical morphology toES-derived NS lines, expressed neural precursor cell/radial glial markerimmunoreactivity, and could differentiate into neurons and astrocytes.This showed that the NS cells obtained from fresh foetal neurosphereswere the same as those from neurospheres frozen down and then grown asneurospheres for a long time.

Example 8

LC1 mouse NS cells were transplanted into foetal rat brain. Confocalimages of NS cells, lenti-virally transduced with eGFP, were taken oneweek after transplantation into the ventricle of E14.5 rats. Donor cellsmigrated from the ventricle into the parenchyma in clusters and assingle cells. Grafted cells showed co-localization of eGFP and theneuronal marker MAP2, the astroglia marker GFAP or the progenitor cellmarker nestin. Thus, the NS cells migrated and differentiated aftertransplantation in the foetal rat brain.

Example 9

Protocols For Derivation and Manipulation of NS Cell Lines

We devised the following protocols for derivation and manipulation of NScell lines.

Derivation of Mouse NS Cell Lines from ES Cells

ES cells can efficiently be converted to Sox1 expressing neuralprogenitors in adherent monolayer culture [P1]. Detailed protocols andtroubleshooting and for this ES cell differentiation are describedelsewhere [P2]. Briefly, ES cells are routinely cultured underfeeder-free conditions in medium supplemented with 10% foetal calf serumand 100 U/ml recombinant leukaemia inhibitory factor (LIF) ongelatin-coated tissue culture plastic [P3]. Undifferentiated ES cellsare expanded to ˜80% confluence in a T75 flask (Iwaki), trypsinised andresuspended in N2B27 media [P2]. Cells are plated onto 9 cm plates(Iwaki) that have been coated with a 0.1% gelatin solution (Sigma) forat least 10 mins then allowed to dry. As initial plating density is acrucial parameter for efficient neural induction, and can vary betweenES cell lines, we routinely seed several different cell densities(0.8×10⁶, 1×10⁶ and 1.2×10⁶) per plate. Culture medium is changed eachday, in the process removing detached or dead cells. Under theseconditions 50-80% of cells will undergo neural lineage specificationwithin 4-5 days, and with overt neuronal differentiation detectable fromday 5 onwards.

Conversion of heterogeneous progenitor cultures to homogeneous NS celllines can be achieved as follows. Day 7 differentiated cultures aretrypsinised and 2-3×10⁶ cells are re-plated into an uncoated T75 flaskin NS expansion media, comprising NS-A media (Euroclone) supplementedwith L-glutamine, 2 mM final (Gibco), modified N2 supplement (freshlyprepared in house) [P2] and 10 ng/ml of both mouse EGF (Peprotech) andhuman FGF-2 (Peprotech). Expansion media can be stored at 4° C. for upto 4 weeks. Within 2-3 days the flask will contain many thousands offloating aggregates in suspension culture (absolute number variesaccording to efficiency of initial ES cell differentiation). Cellaggregates are harvested by mild centrifugation or allowed to settleunder gravity in a 30 ml universal tube for 10 mins. This step removesdebris and dead cells, thus enriching for NS cell founders, and ensurescomplete media exchange. Cells are replated in 10 ml of fresh NSexpansion medium onto a gelatin coated T75 flask (Iwaki). After afurther 3-7 days, cell aggregates will attach to the flask and shortlythereafter cells outgrow with characteristic bipolar NS cell morphology.Following extensive outgrowth of cells (a further 3-4 days) the entirepopulation is trypsinised and re-plated as single cells onto a gelatincoated T75 flask in expansion medium. Cells grow very rapidly (doublingtime ˜25 hrs) and remain adherent. Within several passages residualdifferentiated and blast cells are eliminated (monitored by GFAP andTuJ1 immunostaining) and cultures are uniformly positive for NS cellmarkers.

For 46C ES cells (Sox1-GFP-IRES-pac knock-in) [P4] a transient selectionwith puromycin (0.5□g/ml) can be used to eliminate non-neural cells andderive NS cells via continuous adherent culture. 46C ES cells (1×10⁶)are plated in N2B27 media onto a gelatin coated 9 cm dish to induceneural commitment. Six days later puromycin is added for 48 hrs. Theenriched Sox1 expressing cell population (˜3-5×10⁶) is then re-plated inN2B27 medium containing EGF (10 ng/ml) and FGF-2 (10 ng/ml) on gelatincoated 9 cm plates. The initially morphologically heterogenous Sox1expressing population progressively acquires a Sox1 negative characterand after 3-4 passages uniform NS cell morphology and marker expression.

Derivation of Mouse NS Cell Lines from Foetal CNS and Neurospheres

Cell clusters form in suspension upon dissociation of foetal E16.5cortex and primary culture in NS cell expansion media. These primaryaggregates can be readily converted into adherent NS cell lines byplating onto gelatin-coated substrate in expansion medium. In order topromote attachment, it is important that debris/dead cells are firsteffectively removed by sedimentation and medium is exchanged completely.Cell aggregates will attach and outgrow over 2-5 days. Outgrowing cellscan subsequently be trypsinised to single cells, re-plated andpropagated in NS cell expansion medium. From passaged foetalneurospheres [P5], NS cells can conveniently be established bydissociation and plating directly on gelatin coated plastic in NSexpansion medium. During the initial few passages the derived NS lineshave a tendency to aggregate, detach from the flask and re-formneurospheres, particularly if the cell density becomes high. Culturesshould therefore be passaged at or below 50% confluence. The tendency tospontaneously aggregate is variable, but is generally reduced uponfurther passaging, or through establishment of clonal cell lines.

Passage and Expansion of NS Cells

Once established, NS cells are propagated in NS expansion media. NScells are grown on gelatin coated plates/flasks and are routinely split1 in 2 to 1 in 5. NS cells have a doubling time of around 25 hrs. Cellsare passaged using trypsin/EDTA or through incubation with calciummagnesium-free PBS (Sigma). For establishment of clonal lines, singlecells can be deposited in gelatin-coated microwells in expansion medium.Less rigorously, cells can be plated at very low density, 1000 cells per9 cm dish. Colonies appear within two weeks and can be picked andexpanded.

Cryopreservation and Recovery of NS Cells

NS cells are readily recovered following freezing/thawing. Routinely wetrypsinise a 60-90% confluent T75 flask, and resuspend the pellet into1.5 ml NS expansion media plus 10% DMSO. This is then split into 3×1 mlcryotubes (Nunc) and stored at −80° C. NS cells are recoverablefollowing more than 6 months storage in these conditions. For long termstorage frozen vials are transferred to liquid nitrogen. NS cells arethawed by rapidly bringing the vial to 37° C. followed by transfer to 10ml of pre-warmed NS expansion media. Cells are pelleted and thenresuspended in fresh expansion media to remove DMSO. Cell recoveryfollowing cryopreservation is >95% for NS cells.

Astrocyte and Neuronal Differentiation of NS Cells

Rapid differentiation of NS cells to GFAP positive astrocytes occurswithin 2 days of exposure of NS cells to BMP4 (10 ng/ml) or 1% FCS inNS-A (with N2, without EGF/FGF) on gelatin coated flasks/plates. Celldensity is not a crucial parameter for astrocyte differentiation.

For neuronal differentiation NS cells are harvested using Accutase(Sigma) or calcium/magnesium-free PBS to detach cells and 0.5-1.0×10⁴cells are re-plated into each well of a poly-ornithine/laminin coated4-well multidish plate (Nunc) in NS-A medium supplemented with FGF-2 (5ng/ml), modified N2, and B27 (Gibco). We find that the NS-A basal mediumis more permissive for neuronal differentiation, than other basal media.A half volume of medium is replaced every 2-3 days to maintainconditioning of medium. After 7 days in these conditions we exchangemedia to NS-A mixed with Neurobasal medium (Gibco) in a ratio of 1:1supplemented with 0.25×N2 plus B27 and without EGF or FGF. Thisformulation promotes further neuronal differentiation and maturation.For longer term cultures of neurons (beyond 14 days) we exchange mediato Neurobasal supplemented with B27 without N2 in the presence of BDNF(10 ng/ml).

Culture and Neuronal Differentiation of Human NS Cells

Human NS cells are expanded on 0.1% gelatine (Sigma) coatedflasks/plates in expansion media as for mouse NS cells additionallysupplemented with 100 U/ml recombinant human leukaemia inhibitory factor(LIF). Cells have a doubling time of approximately 1 week. They arepassaged with trypsin once reaching ˜30% confluence and split 1 in 2.Overgrowth should be avoided to maintain monolayer culture and preventaggregation and detachment.

For neuronal differentiation, human NS cells are harvested usingAccutase (Sigma) and around 1×10⁴ cells re-plated into each well ofpoly-ornithine /laminin (Sigma) coated 12-well plate (Iwaki) inexpansion media. Cells are expanded until they reach around 80%confluent. Neuronal differentiation is induced by removing EGF and LIFfrom expansion media. After 7 days in the absence of EGF and LIF,exchange media to NS-A mixed with Neurobasal media in a ratio of 1:1(Gibco) supplemented with 0.5×N2, B27, FGF2 (5 ng/ml), and BDNF (10ng/ml). After a further 7 days in these conditions, media is switched toNeurobasal media supplemented with B27 and BDNF (10 ng/ml) without N2 orFGF-2. Half the medium is exchanged every 2 or 3 days as for mouse NScells throughout this protocol. After a further 10 days cells ofneuronal morphology immunoreactive with TuJ1 and MAP2 represent up to40% of total cell numbers. Significant numbers of astrocytes are alsogenerated, in contrast to the mouse NS cell protocol for neuronaldifferentiation in which few GFAP positive cells emerge.

The invention thus provides methods and media for obtaining andmaintaining neural stem cells of many species in asymmetrically-dividing, undifferentiated state. The invention has beencarried out using cells obtained or extracted from ES cell, foetal andadult sources. In all cases the resultant cells obtained by followingthe methods described herein look and behave substantially the same;they all can be maintained in high purity cultures over high numbers of,e.g. hundreds of, doublings with a high proportion of the cellsretaining the ability to form neurons and glia. Specifically, NS cellshave been successfully obtained from human (ES, foetal CNS), mouse (ES,foetal CNS, adult CNS) and rat (foetal CNS). Mouse neural stem cellshave been grown in culture in pure populations and after beingtransplanted grew without forming tumours but differentiated in vivo.

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PROTOCOL REFERENCES

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1. A method of promoting symmetrical division of neural stem (NS) cells,comprising culturing said cells attached to a substrate in a mediumcontaining: (a) an activator of a signalling pathway downstream from areceptor of the EGF family; and (b) an activator of a signalling pathwaydownstream from an FGF receptor.
 2. The method of claim 1, wherein theactivator of a signalling pathway downstream from a receptor of the EGFfamily is an agonist of an EGF receptor.
 3. The method of claim 2,wherein the agonist is selected from the group consisting of EGF, TGF-A,amphiregulin, heparin binding-EGF, epiregulin, betacellulin, neuregulin1, neuregulin 2, neuregulin 3, neuregulin 4, and Cripto-1.
 4. The methodof claim 1, wherein the activator of a signalling pathway downstreamfrom an FGF receptor is an agonist of an FGF receptor.
 5. The method ofclaim 4, wherein the agonist is FGF-2.
 6. A method for obtaining neuralstem cells comprising: (1) obtaining a mixed population of cellscontaining a neural stem cell; (2) plating the cells in mediumcomprising (a) an activator of a signalling pathway downstream from areceptor of the EGF family; and (b) an activator of a signalling pathwaydownstream from an FGF receptor; (3) culturing the cells to obtainaggregates of cells; (4) harvesting the aggregates of cells; (5)replating cells from the aggregates of cells in medium comprising (a) anactivator of a signalling pathway downstream from a receptor of the EGFfamily; and (b) an activator of a signalling pathway downstream from anFGF receptor; (6) culturing the cells; (7) harvesting cells from theculture of (6); (8) replating harvested cells from (7) as single cellsin medium containing (a) an activator of a signalling pathway downstreamfrom a receptor of the EGF family; and (b) an activator of a signallingpathway downstream from an FGF receptor.
 7. The method of claim 6,comprising selecting for cells that express a neural stem cell specificmarker.
 8. The method of claim 7, comprising linking expression of theneural stem cell specific marker to a promoter preferentially active inneural stem cells compared to its expression in differentiated progenythereof.
 9. The method of claim 6, comprising passaging the cells at orbelow 65% confluence,
 10. The method of claim 9, comprising passagingthe cells at or below 55% confluence.
 11. A composition, comprising:neural stem cells; an activator of a signalling pathway downstream froma receptor of the EGF family; and an activator of a signalling pathwaydownstream from an FGF receptor.
 12. The composition of claim 11,wherein at least 50% of said neural stem cells aresymmetrically-dividing neural stem cells.
 13. The composition of claim11, wherein at least 70% of said neural stem cells aresymmetrically-dividing neural stem cells.
 14. The composition of claim11, wherein at least 80% of said neural stem cells aresymmetrically-dividing neural stem cells.
 15. The composition of claim11, wherein at least 90% of said neural stem cells aresymmetrically-dividing neural stem cells.
 16. The composition of claim11, wherein at least 95% of said neural stem cells aresymmetrically-dividing neural stem cells.
 17. The composition of claim11, wherein at least 97% of said neural stem cells aresymmetrically-dividing neural stem cells.
 18. The composition of claim11, wherein the neural stem cells are characterized in that they arepositive for the expression of RC2, 3CB2 , BLBP and Sox-2.hh
 19. Thecomposition of claim 18, wherein the neural stem cells are furthercharacterized in that they are positive for the expression of at leastone of (i) GLAST, (ii) Pax-6, (iii) the neural precursor markers nestinor vimentin, (iv) the LewisX antigen, (v) Musashi-1 and (vi) prominin.20. The composition of claim 19, wherein the neural stem cells arefurther characterised in that they are negative for the expression of atleast one of Oct4 and Nanog.
 21. The composition of claim 19, whereinthe neural stem cells are positive for the expression of Sox-2, andnegative for the expression of Sox-1.
 22. The composition of claim 11,wherein no more than 1% of the cells in the composition are positive forthe expression of markers for mature astrocytes, neurons oroligodendrocytes.
 23. A composition comprising neural stem cells,wherein the neural stem cells are in an adherent culture and at least80% of the cells in the composition are neural stem cells.
 24. Thecomposition of claim 23, wherein at least 90% of the cells in thecomposition are neural stem cells.
 25. The composition of claim 23,wherein the neural stem cells have been passaged at least 30 times. 26.The composition of claim 25, wherein the neural stem cells have beenpassaged at least 60 times.
 27. A method of obtaining neurons,comprising (a) culturing neural stem cells in the presence of an agonistof an FGF receptor and in the absence of an agonist of an EGF receptor;and (b) thereafter, culturing the cells in the absence of an agonist ofan FGF receptor and in the absence of an agonist of an EGF receptor. 28.The method of claim 27, comprising plating cells in monolayer culture.29. The of claim 27, comprising transferring NS cells to medium free ofEGF but which contains FGF-2 and culturing them for a period of at least2 days and them removing FGF-2 from the medium.
 30. The method of claim27, comprising obtaining neural stem cells according to the method ofclaim
 6. 31. The method of claim 27, comprising culturing thecomposition of claim
 13. 32. The method of claim 27, comprisingculturing the composition of claim
 14. 33. The method of claim 27,comprising culturing the composition of claim
 15. 34. Neurons obtainedby the method of claim 31.